The Use of a Dimeric Four-helix Bundle as a Model to Study Protein Folding and Design

Abstract

Fifty years after the work of Anfinsen, we are still unable to definitively determine the tertiary structure of proteins from their sequences. While it is true that recent computational studies have shown promising results, it is still very difficult to conclude whether a given sequence will fold at all, let alone predict its conformation. Experimental approaches to the protein folding problem allow us to improve upon computational models by obtaining physical data on how specific residue mutations affect structure, function, and stability. The four-helix bundle rop was chosen for our studies. Rop is a small protein with an abundance of high-resolution data, making it an ideal choice for site-directed mutagenesis experiments. Additionally, an in vivo screen which uses GFP as a reporter exists for rop. We first set out to create a cysteine-free variant of rop that behaves like the wild-type in structure, function, and stability. After testing several mutants that replace the two cysteines with similar residues, it was found that the A38/V52 variant was functionally active, expressed well in solution, and possessed a high degree of α-helical content. 15N-HSQC NMR data of this A38/V52 mutant showed a peak dispersion that was very similar to wild-type. Preliminary x-ray crystallographic data indicates that this cys-free mutant deviates from wild-type by 0.37 Å. A new method for purifying and screening protein library variants on a high-throughput scale was developed. The purification method takes advantage of recent developments in high-throughput protein manipulation tools, and the screening method is able to inexpensively screen almost one hundred variants at once for stability. Two single-chain rop constructs were designed with the intent to create a single-chain monomer that mimicked the structure and function of wild-type rop, which is normally a dimer. Both constructs were found to be functionally active. Further characterization is in progress.